Methadone has been widely used as an aid in withdrawal from heroin addiction. Compliance with methadone therapy is frequently monitored by analysis of urine samples for the presence of methadone, which can be performed using one of several commercially available immunoassays for methodone.
However, there are several occasions when a simple assay for methadone provides in incorrect or incomplete diagnostic information. For example, the methadone may be so extensively metabolized that the concentration excreted falls below that of the assay being used. To the extent that the assay distinguishes between methadone and excreted metabolites, the test can be negative even for a patient in full compliance with therapy (Nicar et al., Clin. Chem. 42:543, 1996). In another example, a patient may add methadone to their sample to disguise the fact that they are not adhering to the treatment protocol. Samples that have been tampered with can in principle be distinguished in that they will not contain filterable metabolites of methadone that are also present in urine when the methadone treatment protocol is being properly adhered to.
Both these types of situations could be easily recognized if it were possible to independently measure the presence of the excreted metabolite. In practical terms, detecting methadone metabolites is difficult because of the chemical and immunological similarity with methadone itself. Accordingly, there is a need for reagents and techniques that would permit routine monitoring of methadone metabolites in a clinical setting.
A major pathway of metabolism of methadone is demethylation to the presumed intermediate N-desmethylmethadone, leading to urinary excretion of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine herein abbreviated as EDDP), 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP), and their ring-hydroxylated analogs (Sullivan et al., J. Medicinal Chem. 16:909, 1973). These structures are depicted in FIG. 1. Lesser amounts of 4-dimethylamino-2,2-diphenylvaleric acid (formed by side-chain oxidation); 1 ,5-dimethyl-3,3-diphenyl-2-pyrrolidone (resulting from subsequent N-demethylation and cyclization), ring-hydroxylated methadone, and normethadol are also found. Methadone N-oxide is formed by storage of samples at 30.degree. C. In human volunteers receiving.about.100 mg of dl-methadone orally, EDDP was found by Sullivan et al. to be the most prominent metabolite in urine, present at a level roughly comparable with that of methadone itself.
In order to detect methadone metabolites, Sullivan et al. extracted urine with methylene chloride, hydrolyzed the reconstituted extract using .beta.-glucuronidase and aryl sulfatase, optionally acetylated or methylated the product, and then characterized it by gas chromatography or combined gas chromatography-mass spectroscopy.
The synthesis and spectral properties of optically active EDDP were described by Brine et al. (J. Heterocyclic Chem., 23:369, 1986). The .sup.1 H--NMR analysis suggested that the free base exists predominantly in an enamine form. CD and ORD studies provided data consistent with a fairly rigid enamine structure.
Simultaneous gas chromatography mass spectrometry (GC/MS) assay for EDDP in urine was described by Baugh et al. (J. Forensic Sci. 36:548, 1991). Urine was extracted with 1-chlorobutane at pH.about.9, the organic phase was back-extracted into acetate buffer, adjusted to pH.about.9, and re-extracted with I -chlorobutane. Area corresponding to ions at m/z 277, 262, and 276 was measured. Quantitation was enhanced by using deuterated methadone as the internal standard. Although the assay is quantitative, it relies on a multi-step extraction procedure and the availability of equipment to perform GC/MS.
Thin-layer chromatography (TLC) screening of methadone and EDDP in urine was described by Budd et al. (Clin. Toxicol. 16:55, 1980). Metabolites were extracted into an organic solvent, and then chromatographed in ethyl acetate:methanol:diethylamine or ethyl acetate:methylene choride:propylamine. Dried plates were developed using acidified iodoplatinate reagent. Although this permits a number of samples to be processed in the same day, this type of assay is non-quantitative and subject to variations in solvent mixtures.
More suited for routine clinical analysis are immunoassays, in which a specific antibody is used to distinguish and quantify an analyte of interest in a biological sample. The general art of immunoassay and its use in clinical monitoring is well known. Assays for analytes of the size of EDDP are often competition assays. Immunoassays typically involve either the specific isolation of the analyte from the sample mediated by antibody, or the formation of analyte-antibody complexes in situ (a "homogeneous" assay system). In either case, formation of an analyte-antibody complex ultimately leads to a signal which is directly or inversely related to the amount of analyte present in the original sample.
A particularly powerful homogeneous assay system is the cloned enzyme donor immunoassay (CEDIA.RTM.), described in U.S. Pat. No. 4,708,929, and in Henderson, Clin. Chem. 32:1637, 1986. In a preferred form of the CEDIA.RTM. assay, two subunits of the enzyme .beta.-galactosidase associate to provide the detectable signal, which is quantitatively affected by analyte-specific antibody except in the presence of a sample containing free analyte.
All specific immunoassays require the availability of an antibody that binds the analyte of interest but not potential interfering substances. An immunoassay for EDDP capable of differentiating samples spiked with methadone requires an antibody with a very high relative affinity for EDDP in relation to methadone.
A commercial assay for EDDP is marketed by Diagnostic Reagents, Inc. of Mountain View, Calif. This is a homogeneous enzyme immunoassay based on the binding of an anti-EDDP antibody with a glucose-6-phosphate dehydrogenase drug conjugate, which inhibits the activity of the enzyme. Presence of sample analyte in the reaction mixture binds the antibody and increases enzyme activity. The utility of this assay is limited by its specificity. EDDP gives a positive result at 300 ng/mL, EMDP at 400 ng/mL, and methadone itself at 5000 ng/mL. In other words, the assay when used alone is unable to distinguish between a sample containing EDDP, and another sample spiked with methadone at a somewhat higher level.
The limited specificity in current art EDDP assays is attributable to the limited specificity in the antibody in the assay. This in turn is attributable to the unsuitability of current art immunogens for generating antibodies with better specificity.